PROJECT SUMMARY ABSTRACT Angiogenesis, or formation of new vasculature from pre-existing vessels, is a complex process coordinated by multiple cell types including endothelial (EC) and smooth muscle (SM) cells of the vessel wall. Notch signaling is an evolutionarily conserved pathway required in ECs for developmental, reparative and pathological angiogenesis and collaborates with vascular endothelial growth factor (VEGF)-dependent signaling to optimize organization and competency of neovasculature. These signals in ECs are especially relevant in contexts of pathological hypoxia induced by ischemia or hypoxemia, wherein low tissue oxygen levels trigger hypoxia inducible factor (HIF) transcriptional activity governing production of critical angiogenic factors including VEGF. Despite their juxtaposition to ECs within the vessel wall, much less is known of the contribution of SM to the angiogenic process in health or disease. We have previously shown that heterotypic cell interactions support instructional signaling between SM Notch ligands and EC Notch receptors. These contacts modulate EC gene expression and function and mice deficient in SM Notch signaling display impaired physiological angiogenesis. We now suggest that SM Notch signaling is required for adaptive angiogenesis induced by pathological hypoxia. We have identified a functional synergism between Notch and HIF signaling pathways in SM under hypoxic conditions and demonstrate a reciprocal dependence on target gene expression including the augmentation of Notch ligand and VEGF. Therefore, Notch signaling in SM appears necessary for HIF activity induced by hypoxia. These findings further suggest that enhanced Notch ligand and SM-derived VEGF act as juxtacrine and paracrine influences on EC behavior. Hence, the major themes in this project proposal include the identification of molecular mechanisms in SM that underlie functional synergism between Notch and HIF signaling and the characterization of hypoxic vascular SM impact on angiogenesis. In Aim 1, we interrogate molecular crosstalk between components of Notch and HIF pathways. Aim 2 measures EC responses in co-culture with select Notch/HIF-deficient SM. Aim 3 characterizes angiogenic capacity in animals with combined deficiency in SM Notch/HIF through distinct models of pathologically induced hypoxia. Together, our experimental approaches include a full spectrum of in vivo, ex vivo and in vitro vascular analyses necessary for understanding novel mechanisms of angiogenic control.